The Experts below are selected from a list of 1410 Experts worldwide ranked by ideXlab platform
Hiroshi Nikaido - One of the best experts on this subject based on the ideXlab platform.
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aminoacyl β naphthylamides as substrates and modulators of acrb multidrug efflux pump
2016Co-Authors: Alfred Dieudonné Kinana, Attilio Vittorio Vargiu, Hiroshi NikaidoAbstract:Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of Nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the Nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.
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Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli
2015Co-Authors: Ketaki Soparkar, Alfred Dieudonné Kinana, Jon W. Weeks, Keith D. Morrison, Hiroshi Nikaido, Rajeev MisraAbstract:ABSTRACT The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The Nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored Nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N -phenyl-1-naphthylamine, the TM suppressors did not improve the Nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.
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mechanism of recognition of compounds of diverse structures by the multidrug efflux pump acrb of escherichia coli
2010Co-Authors: Yumiko Takatsuka, Cheng Chen, Hiroshi NikaidoAbstract:The AcrB trimeric multidrug efflux transporter of Escherichia coli pumps out a very wide spectrum of compounds. Although minocycline and doxorubicin have been cocrystallized within the large binding pocket in the periplasmic domain of the binding protomer, nothing is known about the binding of many other ligands to this protein. We used computer docking to evaluate the interaction of about 30 compounds with the binding protomer and found that many of them are predicted to bind to a narrow groove at one end of the pocket whereas some others prefer to bind to a wide cave at the other end. Competition assays using Nitrocefin efflux and covalent labeling of Phe615Cys mutant AcrB with fluorescein-5-maleimide showed that presumed groove-binders competed against each other, but cave-binders did not compete against groove-binders, although the number of compounds tested was limited. These results give us at least a hypothesis to be tested by more biochemical and genetic experiments in the future.
Alfred Dieudonné Kinana - One of the best experts on this subject based on the ideXlab platform.
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aminoacyl β naphthylamides as substrates and modulators of acrb multidrug efflux pump
2016Co-Authors: Alfred Dieudonné Kinana, Attilio Vittorio Vargiu, Hiroshi NikaidoAbstract:Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of Nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the Nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket.
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Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli
2015Co-Authors: Ketaki Soparkar, Alfred Dieudonné Kinana, Jon W. Weeks, Keith D. Morrison, Hiroshi Nikaido, Rajeev MisraAbstract:ABSTRACT The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The Nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored Nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N -phenyl-1-naphthylamine, the TM suppressors did not improve the Nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.
Michael W. Crowder - One of the best experts on this subject based on the ideXlab platform.
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Structural and Kinetic Studies on Metallo-β-lactamase IMP-1
2016Co-Authors: Dionne H. Griffin, Robert M Breece, Brian Bennett, David L Tierney, Timothy K. Richmond, Carlo Sanchez, Abraham Jon Moller, Michael W. CrowderAbstract:In an effort to probe for metal binding to metallo-β-lactamase (MβL) IMP-1, the enzyme was overexpressed, purified, and characterized. The resulting enzyme was shown to bind 2 equiv of Zn(II), exhibit significant catalytic activity, and yield EXAFS results similar to crystallographic data previously reported. Rapid kinetic studies showed that IMP-1 does not stabilize a Nitrocefin-derived reaction intermediate; rather, the enzyme follows a simple Michaelis mechanism to hydrolyze Nitrocefin. Metal-substituted and metal-reconstituted analogues of IMP-1 were prepared by directly adding metal ion stocks to metal-free enzyme, which was generated by dialysis versus EDTA. UV–vis studies on IMP-1 containing 1 equiv of Co(II) showed a strong ligand-to-metal charge transition at 340 nm, and the intensity of this feature increased when the second equivalent of Co(II) was added to the enzyme. EXAFS fits on IMP-1 containing 1 equiv of Co(II) strongly suggest the presence of a metal–metal interaction, and EPR spectra of the IMP-1 containing 1 and 2 equiv of Co(II) are very similar. Taken together, steady-state kinetic and spectroscopic studies suggest that metal binding to metal-free IMP-1 follows a positive-cooperative mode
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motion of the zinc ions in catalysis by a dizinc metallo beta lactamase
2009Co-Authors: Robert M Breece, Zhenxin Hu, Michael W. Crowder, Brian Bennett, David L TierneyAbstract:We report rapid-freeze-quench X-ray absorption spectroscopy of a dizinc metallo-beta-lactamase (MbetaL) reaction intermediate. The Zn(II) ions in the dinuclear active site of the S. maltophilia Class B3 MbetaL move away from each other, by approximately 0.3 A after 10 ms of reaction with Nitrocefin, from 3.4 to 3.7 A. Together with our previous characterization of the resting enzyme and its Nitrocefin product complex, where the Zn(II) ion separation relaxes to 3.6 A, these data indicate a scissoring motion of the active site that accompanies the ring-opening step. The average Zn(II) coordination number of 4.5 in the resting enzyme appears to be maintained throughout the reaction with Nitrocefin. This is the first direct structural information available on early stage dizinc metallo-beta-lactamase catalysis.
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role of the zn1 and zn2 sites in metallo beta lactamase l1
2008Co-Authors: Gopalraj Periyannan, Brian Bennett, Michael W. CrowderAbstract:In an effort to probe the role of the Zn(II) sites in metallo-beta-lactamase L1, mononuclear metal ion containing and heterobimetallic analogues of the enzyme were generated and characterized using kinetic and spectroscopic studies. Mononuclear Zn(II)-containing L1, which binds Zn(II) in the consensus Zn1 site, was shown to be slightly active; however, this enzyme did not stabilize a Nitrocefin-derived reaction intermediate that had been previously detected. Mononuclear Co(II)- and Fe(III)-containing L1 were essentially inactive, and NMR and EPR studies suggest that these metal ions bind to the consensus Zn2 site in L1. Heterobimetallic analogues (ZnCo and ZnFe) analogues of L1 were generated, and stopped-flow kinetic studies revealed that these enzymes rapidly hydrolyze Nitrocefin and that there are large amounts of the reaction intermediate formed during the reaction. The heterobimetallic analogues were reacted with Nitrocefin, and the reactions were rapidly freeze quenched. EPR studies on these samples demonstrate that Co(II) is 5-coordinate in the resting state, proceeds through a 4-coordinate species during the reaction, and is 5-coordinate in the enzyme-product complex. These studies demonstrate that the metal ion in the Zn1 site is essential for catalysis in L1 and that the metal ion in the Zn2 site is crucial for stabilization of the Nitrocefin-derived reaction intermediate.
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KINETIC MECHANISM OF METALLO-BETA -LACTAMASE L1 FROM STENOTROPHOMONAS MALTOPHILIA
1999Co-Authors: Silvia Mcmanus-munoz, Michael W. CrowderAbstract:The reaction of Nitrocefin with metallo-β-lactamase L1 from Stenotrophomonas maltophilia was studied using rapid-scan and stopped-flow ultraviolet−visible (UV−vis) studies in an effort to discern the kinetic mechanism used by L1 to hydrolyze penicillins and cephalosporins. Rapid-scan and stopped-flow UV−vis studies of Nitrocefin hydrolysis by L1 identified three species: (1) the substrate (Nitrocefin) displayed an absorbance peak at 390 nm (e = 11 500 M-1 cm-1) that decreased during the reaction with a rate constant of 170 ± 30 s-1; (2) the product (hydrolyzed Nitrocefin) displayed an absorbance peak at 485 nm (e = 17 420 M-1 cm-1) that increased during the reaction with rate constant of 40 ± 1 s-1; and (3) an intermediate displayed an absorbance peak at 665 nm (e = 32 000 M-1 cm-1) that increased initially with a rate constant of 190 ± 3 s-1 and then decreased with a rate constant of 38 ± 2 s-1. Single-turnover experiments demonstrated that there were no pre-steady-state bursts in the reaction of L1 wit...
Jan Tommassen - One of the best experts on this subject based on the ideXlab platform.
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antibiotic trapping by plasmid encoded cmy 2 β lactamase combined with reduced outer membrane permeability as a mechanism of carbapenem resistance in escherichia coli
2013Co-Authors: Wil H F Goessens, Akke K Van Der Bij, Ria Van Boxtel, Johann D D Pitout, Peter Van Ulsen, Damian C Melles, Jan TommassenAbstract:A liver transplant patient was admitted with cholangitis, for which meropenem therapy was started. Initial cultures showed a carbapenem-susceptible (CS) Escherichia coli strain, but during admission, a carbapenem-resistant (CR) E. coli strain was isolated. Analysis of the outer membrane protein profiles showed that both CS and CR E. coli lacked the porins OmpF and OmpC. Furthermore, PCR and sequence analysis revealed that both CS and CR E. coli possessed bla(CTX-M-15) and bla(OXA-1). The CR E. coli strain additionally harbored bla(CMY-2) and demonstrated a >15-fold increase in β-lactamase activity against Nitrocefin, but no hydrolysis of meropenem was detected. However, Nitrocefin hydrolysis appeared strongly inhibited by meropenem. Furthermore, the CMY-2 enzyme demonstrated lower electrophoretic mobility after its incubation either in vitro or in vivo with meropenem, indicative of its covalent modification with meropenem. The presence of the acyl-enzyme complex was confirmed by mass spectrometry. By transformation of the CMY-2-encoding plasmid into various E. coli strains, it was established that both porin deficiency and high-level expression of the enzyme were needed to confer meropenem resistance. In conclusion, carbapenem resistance emerged by a combination of elevated β-lactamase production and lack of porin expression. Due to the reduced outer membrane permeability, only small amounts of meropenem can enter the periplasm, where they are trapped but not degraded by the large amount of the β-lactamase. This study, therefore, provides evidence that the mechanism of "trapping" by CMY-2 β-lactamase plays a role in carbapenem resistance.
Alain Hartmann - One of the best experts on this subject based on the ideXlab platform.
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a Nitrocefin based amperometric assay for the rapid quantification of extended spectrum β lactamase producing escherichia coli in wastewaters
2017Co-Authors: Benoît Chantemesse, Laetitia Betelli, Alain Hartmann, Fabienne Vienney, Sébastien Solanas, Loic Bollache, Murielle RocheletAbstract:Abstract A sensitive and inexpensive amperometric assay based on the electrochemical detection of the β-lactamase activity using the Nitrocefin as substrate was developed for the rapid and quantitative detection of extended spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) in urban wastewaters. The specific detection of ESBL-EC was achieved by culturing the filtered sample in a medium containing the cefotaxime supplemented or not with the potassium clavulanate inhibitor. This step was followed by the incubation of each subculture filtrate with the Nitrocefin substrate which hydrolysis was monitored by amperometry using disposable carbon screen-printed sensors. Current intensities iCef and iClav correspond to the intensity of the anodic current measured (∼+ 0.2 V vs. Ag/AgCl) for the sample incubated with the cefotaxime without and with potassium clavulanate, respectively. The intensity value i = iCef – iClav was chosen as the analytical response. ESBL-EC calibration plots were established with artificially contaminated wastewater samples. This assay allowed the detection of ESBL-EC amounts as low as 10 cfu in treated effluents and 100 cfu in raw wastewaters with short time analysis of 5.5 h and 4.5 h, respectively. The amperometric method was applied to the analysis of 38 wastewater samples and the results were in good agreement with CFU counts on a selective chromogenic medium for 24 h. Owing to its rapidity, convenience, low-cost and portability, this assay is a promising tool to obtain quantitative data on antimicrobial-resistant E. coli in wastewater effluents. Furthermore, this assay might be used to improve wastewater treatment plant processes in order to minimize the release of antibiotic resistant bacteria into the aquatic environment.
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An amperometric method for the rapid detection of extended-spectrum β-lactamase producing Escherichia coli in wastewater treatment plant effluents
2016Co-Authors: Benoît Chantemesse, Laetitia Betelli, Alain Hartmann, Fabienne Vienney, Murielle Déquaire-rocheletAbstract:Context: Extended-spectrum β-lactamase-producing Escherichia coli (ESBL E. coli) are resistant to most β-lactams and have become a major concern in human and veterinary medicine. As E. coli and antibiotic resistant strains are part of the intestinal flora of humans, large amounts of these bacteria are present in wastewaters. Though treatments are performed in wastewater treatment plants (WWTP), large quantities of bacteria are still present in the treated effluents rejected into the environment. These releases can cause contaminations of recreational waters and thus present a health risk to exposed populations. Therefore, rapid and convenient assays are highly desired for the quantification of ESBL E. coli in the wastewater network and in natural environments. Objective of the study: Development of a Nitrocefin-based amperometric method for the rapid quantification of ESBL E. coli in WWTP effluents Methods: Raw and treated wastewaters were filtered in duplicate through 0.45 μm filters (HAWP, 47 mm, Millipore). The amperometric assay involved two main steps: (1) the subculturing of the filtered samples in the presence of cefotaxime supplemented or not with the potassium clavulanate (ESBL inhibitor) for a few hours (4-5h) followed by, (2) the incubation of each subculture filtrate (v = 10 mL; HVLP filter, 0.45 μm, 13 mm, Millipore) with the Nitrocefin substrate which hydrolysis was monitored by amperometry. iCef and iClav correspond to the intensity of the anodic current measured (~ + 0.2 V vs. Ag/AgCl) for the sample incubated with the cefotaxime without and with potassium clavulanate, respectively. The value i = iCef – iClav was calculated and selected as the analytical response to assess the amount of EBSL E. coli producers. Results: The mean calibration plots for the raw and treated wastewaters (Figure 1) were obtained by analyzing CTX-M type ESBL E. coli strains found in wastewaters (blaCTX-M-1 and blaCTX-M-15 genes) and were used for the determination of ESBL E. coli in 20 raw wastewater and 20 treated wastewater samples. To check the reliability of the amperometric assay, the results were compared to a conventional counting on TBX agar plates supplemented with cefotaxime (Figure 2). Conclusion: An excellent correlation was obtained between the amperometric assay and the enumeration. This amperometric assay (5-6h) which is considerably less time-consuming than the culture-based method (24h) holds great promise for the rapid quantification of ESBL E. coli in the wastewater networks but also in other types of water samples (rivers, marine waters, etc.).
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Amperometric detection of extended-spectrum β-lactamase activity : application to the characterization of resistant E.coli strains
2015Co-Authors: Murielle Déquaire-rochelet, Laetitia Betelli, Fabienne Vienney, Sébastien Solanas, Catherine Neuwirth, Alain HartmannAbstract:The amperometric detection of extended-spectrum β-lactamase (ESBL) with carbon screen-printed sensors was investigated in the presence of the Nitrocefin, a commercially-available β-lactamase chromogenic cephalosporin substrate. Using an ESBL isolated from a clinical sample, it was shown for the first time that the intensity of a specific anodic pic current (EP = [similar]+0.3 V vs. Ag/AgCl) resulting from the catalytic hydrolysis of the β-lactam ring was proportional to the amount of ESBL. The proof-of-principle of a novel susceptibility assay for the rapid and accurate identification of ESBL- producing bacteria was then demonstrated. The detection scheme relied on (i) the culture of the sample in a medium containing the cefotaxime supplemented or not with the clavulanic acid inhibitor to allow the specific determination of ESBL producers (ii) followed by the incubation of the bacteria with the Nitrocefin and (iii) the measurement of the enzyme product by cyclic voltammetry. The amperometric assay was further applied to the characterization of E. coli strains and to the quantification of the ESBL producers. A detection limit of 5 × 104 cfu mL−1 ESBL-producing E. coli was achieved after a 10 min incubation time. In contrast to the approved routine assays, the electrochemical approach, which did not require isolated colonies to be performed, provided quantified results regarding ESBL activity within a few hours. Finally, owing to its cost-effectiveness, portability and simplicity, this test holds great promise for clinical and environmental applications.